Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.
Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.
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DOI：
10.1083/jcb.133.3.559
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发表时间：
1996-05
期刊：
影响因子：
--
通讯作者：
White JM
中科院分区：
文献类型：
--
作者：
Danieli T;Pelletier SL;Henis YI;White JM
在这项研究中，我们测试的假设，流感病毒血凝素（HA）介导的融合是一个合作的事件。为此，我们表征了在9种不同的限定表面密度下表达HA的3 T3细胞系。HA密度范围为1.0至12.6 × 10（3）HA三聚体/微米2，通过定量荧光抗体结合测定。HA的横向移动性和移动的百分比分数在这些细胞中没有显著变化，HA表达细胞和靶RBC之间的接触面积也没有显著变化。使用十八烷基罗丹明（R18）标记的RBC的荧光去猝灭测定分析每个HA表达细胞系的融合反应。对于每个细胞系，我们测量融合开始前的滞后时间、融合的初始速率和融合的最终程度。所有细胞系的最终融合程度相似，并且作为HA表面密度的函数的融合的初始速率显示Michaelis-Menten型依赖性。然而，HA表面密度的融合开始前的滞后时间的依赖性是明显的S形。动力学分析的倒数滞后时间与HA表面密度的数据，由两个日志/日志图和希尔图，表明所观察到的sigmoidicity不反映协同性的HA聚集体的形成水平作为融合的先决条件。相反，在滞后时间期间发生的过程的协同性在稍后的步骤中出现，并且涉及最少三个，并且最可能是四个HA三聚体。提出了一个模型来解释HA融合过程中的协同性。
In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA- expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a Michaelis-Menten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.